siRNA sequence-independent modification formats for reducing off-target phenotypic effects in RNAI, and stabilized forms thereof

Abstract

Modification formats having modified nucleotides are provided for siRNA. Short interfering RNA having modification formats and modified nucleotides provided herein reduce off-target effects in RNA interference of endogenous genes. Further modification formatted siRNAs are demonstrated to be stabilized to nuclease-rich environments. Unexpectedly, increasing or maintaining strand bias, while necessary to maintain potency for endogenous RNA interference, is not sufficient for reducing off-target effects in cell biology assays.

The invention claimed is: 
     
       1. A short interfering RNA, comprising:
 a passenger (sense) oligonucleotide having a length of 17 to 30 nucleotides and comprising: sequence-independent modification format (4):
 (4) 5′ m p -N x -m-m-N q -n r  3′ wherein when p is 0, x is 12; when p is 1, x is 11; when p is 3, x is 9, when p is 4, x is 8; r is 0, 1, or 2; and q is an integer representing the length of the passenger strand minus (p+x+r+2); 
 and 
 
 a guide (antisense) oligonucleotide having 17 to 30 nucleotides, a region of continuous complementarity to the passenger (sense) oligonucleotide of at least 12 nucleotides; the guide strand further having complementarity to at least a portion of target mRNA; 
 wherein 
 each m is independently a bicyclonucleotide, a tricyclonucleotide, or a 2′-modified nucleotide; 
 each n is independently a deoxynucleotide, modified nucleotide, or ribonucleotide and is an overhanging nucleotide; 
 when m is a bicyclonucleotide, each N is independently a nucleotide other than a bicyclonucleotide, 
 when m is a tricyclonucleotide, each N is independently a nucleotide other than a tricyclonucleotide, and 
 when m is a 2′-modified nucleotide, each N is independently a nucleotide other than a 2′-modified nucleotide. 
 
     
     
       2. A composition comprising the short interfering RNA of  claim 1  and a biologically acceptable carrier. 
     
     
       3. The short interfering RNA of  claim 1  wherein at least one internucleoside linkage is other than a phosphodiester internucleoside linkage. 
     
     
       4. The short interfering RNA of  claim 1  wherein the short interfering RNA is further associated with a cell-targeting ligand. 
     
     
       5. The short interfering RNA of  claim 1  wherein a 5′-end is further derivatized with a phosphate, C 1-12 -alkyl, C 1-12 -alkylamine, C 1-12 -alkenyl, C 1-12 -alkynyl, C 1-12 -cycloalkyl, C 1-12 -aralkyl, aryl, acyl, or silyl substituent. 
     
     
       6. A kit comprising the short interfering RNA of  claim 1  and a transfection agent. 
     
     
       7. A method of minimizing off-target events for inhibition of expression of a target gene by RNA interference, comprising: contacting a cell containing the target gene with the short interfering RNA of  claim 1  in an amount sufficient to reduce off-target events while maintaining potency. 
     
     
       8. The method of  claim 7  wherein the 2′ modification of the 2′-modified nucleotide comprises H, bromo, chloro, iodo, fluoro, fluoro in an arabinose conformation (FANA), SH, NH 2 , CN, azide, or OR, R, SR, NHR, or N(R) 2  wherein R is alkyl, alkenyl, alkynyl, alkoxy, oxyalkyl, alkoxyalkyl, or alkylamine where the alkyl portion is C1-C6.